SUMMARY Genetic and cell biological evidence from many laboratories implicates both the normal function and the dysfunction of the presenilins in the fundamental mechanism of Alzheimer's disease. Progressive accumulation of A? appears to begin years before other important pathogenic features of AD such as neuroinflammation and tau tangle formation, and the presenilin/?-secretase complex mediates the final cleavages of APP which control the A?42/43 to 40 ratios that help dictate a person's lifelong propensity to AD. A failed clinical trial of a non-selective ?-secretase inhibitor (semagacestat) has led some to suggest that ?-secretase may no longer be a worthy target for therapeutic development, but we and others (e.g., DeStrooper, Cell, 2014) believe otherwise and seek much deeper knowledge of the presenilin cleavage mechanism -- in order to renew interest in targeting ?-secretase to prevent AD. Indeed, several classes of ?-secretase modulators (as opposed to inhibitors) have been described, and just a few have begun to enter clinical trials. Project 1's long-standing interest in presenilin biology has led us to publish during the current grant period several novel findings about ?-secretase: a) the existence of a physiological ?/?-secretase complex in cells; b) pinpointing the function of Nicastrin as a gatekeeper -- sterically hindering the processing of long substrates; and c) a detailed analysis of presenilin/APP TMD interactions and ?-processivity that explains the reason for the tri-peptide cleavages made by PS. Now, we will build on these advances to delve in further molecular detail into the basic mechanisms of wild-type and FAD mutant presenilin, into how and where certain ?-modulators allosterically influence their processivity, and into the cell biology of a normal ?/?-secretase complex we recently discovered that could be central to AD pathogenesis. We will pursue 3 interrelated but non-overlapping Specific Aims to gather this new knowledge. First, we will use a novel strategy (emerging from our most recent paper ? Bolduc et al, eLife, 2016) to analyze systematically many but not all familial AD mutations in PS1 to learn which PS1 amino acids contribute to the unusual active site that dictates the canonical tripeptide cleavage mechanism of ?- secretase. Second, we will use these FAD mutants to examine both the mechanisms and the PS1 binding sites of some of the most promising GSMs (?-secretase modulators), which are highly attractive candidates for slowing or preventing AD. Third, we will confirm and then functionally analyze an unexpected complex we recently discovered between the two key enzymes that make A? peptides: ?- and ?-secretase. Based on progress in the current grant period and extensive Preliminary Data herein, we are committed to using advanced cell biological and biochemical methods which we are deeply familiar with to elucidate the normal physiology and pathobiological role in AD of one of the most unusual and fascinating protein machines in metazoans: the presenilin/?-secretase complex.